J. Agric. Food Chem. lB81, 29, 653-657
which supports R1= C3H7and R2 = C4Hp This analogy may be applied to the remainder of the CI mass spectra for these alkaloids. Since no CI study was made on the epimeric species of these compounds, it is unknown if the observed differences among the a-and @-ergosine,ergoptine, and ergokrystine (Table I) may be used to differentiate the alkyl moieties when = isobutyl and/or sec-butyl. Also, it is unknown at present if the differences in ion intensities for C’ (Table I) is a reflection of the stability and/or ease of formation of C relative to the substituents R1and h.However, the data indicate that CI mass spectrometry is an effective and useful complement to the E1 spectra for the identification of the ergot peptide alkaloids. ACKNOWLEDGMENT
We thank P. A. Stadler of Sandoz Ltd., Basel, Switzerland, for his generous supply of the ergot cyclol alkaloids and especially for his discussions and information concerning the pyroergopeptine structures. Also, we thank H.G. Floss, Purdue University, West Lafayette, IN, for discussions and suggestions concerning these studies. We also thank John McGuire, Environmental Protection Agency, Athens, GA, and C. Dewitt Blanton, Jr., Medicinal Chemistry Department, University of Georgia School of Pharmacy, Athens, GA, for their valuable comments and criticisms on the manuscript. J. S. Robbins is acknowledged for his invaluable technical assistance during the course of this study. LITERATURE CITED Arsenault, G. P. In “Biomedical Application of Mass Spectrometry”; Waller, G. R., Ed.; Wiley-Interscience: New York, 1972. Bacon, C. W.; Porter, J. K.; Robbins, J. D.; Luttrell, E. S. Appl. Environ. Microbiol. 1977,34, 576.
659
Barber, M.; Weisbach, J. A.; Douglas, B.; Dudek, G. 0. Chem. Znd. (London) 1965, 1072. Belzecki, C. M.; Quigley, F. R.; Floss, H. G.; Crespi-Perellino, N.; Guicciardi, A. J. Org. Chem. 1980,45, 2215. Berde, B.; Schild, H. 0. “Ergot Alkaloids and Related Compounds”; Springer-Verlag: Berlin, Heidelberg, and New York, 1978; p 34. BovB, F. J. “The Story of Ergot”;S. Karger: Basel, Switzerland, 1970; p 42. Brunner, R.; Stiitz, P. L.; Tscherter, H.; Stadler, P. A. Can. J. Chem. 1979,57,1638. Burfening, P. J. J. Am. Vet. Med. Assoc. 1973, 163, 1288. Fales, H. M.; Lloyd, H. A.; Milne, G. W. A. J. Am. Chem. SOC. 1970,92, 1590. Floss, H. G. Tetrahedron 1976,32,873. Inoue, T.;Nakahara, Y.; Niwaguchi, T. Chem. Phurm. Bull. 1972, 20, 409. Kohlmeyer, J.; Kohlmeyer, E. Mycologia 1974,66, 77. Porter, J. K.; Bacon, C. W.; Robbins, J. D. J.Agn’c. Food Chem. 1979,27, 595, and references cited therein. Schmidt, J.; Kraft,R.; Voight, D. Biomed. Moss Spectrom. 1978, 5 , 674. Sprague, R. “Diseases of Cereals and Grasses in North America (Fungi except Smut and Rusts)”; The Ronald Press Co.: New York, 1950. Stadler, P. A,, Sandoz L a . , Basel, Switzerland, personal communication, 1979. Stadler, P. A.; sttitz, P.; Stiirmer, E. Erperientia 1977,33,1552. Voight, D.; Johne, S.; Groger, D. Pharmazie 1974,29, 697. Vokoun, J.; Rehacek, Z.Collect. Czech. Chem. Commun. 1975, 40, 1731. Vokoun, J.; Sajdl, P.; Rehacek, Z. Zentralbl. Bakteriol., Parasitenkd., Infektionskr. Hyg., Abt. 2, Naturwiss.: Allg., Landwirtsch. Tech. Mikrobiol. 1974, 129, 499. Received for review June 30,1980. Revised September 26,1980. Accepted February 9, 1981.
Ergot Alkaloid Identification in Clavicipitaceae Systemic Fungi of Pasture Grasses James K. Porter,* Charles W. Bacon, Joe D. Robbins, and Don Betowski’
The isolation and identification of an alkaloid from Balansia epichl&, Balansia strangulans, and Epichloe typhina that corresponds to 6,7-secoagroclavine (UV; TLC; m / e ) are reported. We report on the production of agroclavine, elymoclavine, penniclavine, and festuclavine by E. typhina. In addition, the two ergot peptide alkaloids from E. typhina previously listed as ergosine and ergosinine when analyzed with isobutane chemical ionization mass spectroscopy corresponded to ergovaline and ergovalinine. Another systemic fungus, Balansia henningsiana, was shown to produce chanoclavine(s), dihydroelymoclavine, and another presently unidentified ergoline alkaloid.
Previous investigations (Bacon et al., 1979; Porter et al., l978,1979a,b) of systemic fungi from toxic pasture grasses established that Balansia epichloe, Balansia claviceps, Toxicology and Biological Constituents Research Unit, Richard B. Russell Agricultural Research Center, Science and Education Administration, U.S. Department of Agriculture, Athens, Georgia (J.K.P., C.W.B., and J.D.R.), and Analytical Chemistry Branch, Environmental Protection Agency, Athens, Georgia (D.B.). ‘Present address: U.S. Environmental Protection Agency, Environmental Monitoring Laboratory, Quality Assurance Division, Las Vegas, NV.
Balansia henningsiana, Balansia strangulans, and Epichloe typhinu produced clavine-type alkaloids in vitro. Bacon et al. (1979) showed that several alkaloids produced by B. epichloe in vitro were also produced in vivo on parasitized smut grass (Sporobolus poiretii). These studies suggest that these systemic grass pathogens should be suspect in ergot toxicity syndromes of cattle and that “ergot” alkaloid biosynthesis occurs in other genera of Clavicipitaceae. The toxins responsible for the “ergot-like” syndromes observed in cattle have not been established. Therefore, it is important to characterize the alkaloids produced by these systemic grass pathogens. Laboratory studies have shown similarities in the capability of Balansia and Epichloe to produce clavine al-
This article not subject to US. Copyright. Published 1981 by the American Chemical Society
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kaloids. The major differences in alkaloid biosynthesis between Balansia and Epichloe is that E. typhina may be distinguished by its ability to produce ergot peptide alkaloids similar to Clauiceps purpurea (Brunner et al., 1979). In addition to the alkaloids that chromatographed with ergosine and ergosinine (Porter et al., 1979a) and chanoclavine I, we also report the identification of agroclavine, elymoclavine, penniclavine, and 6,7-secoagroclavine from a 20-week culture of E. typhina. All of these clavine alkaloids correspond to authentic standards as described (Fehr, 1967; Porter et al., 1978, 1979b). The two ergot peptide fractions labeled ergosinine and ergosine (Porter et al., 1979a) also corresponded with synthetic standards except for the minor discrepancies (i.e,, ion intensities) in the low-resolution mass spectra as reported. These, along with an absence of the molecular ion, the low abundance of the diagnostic fragments of m / e 280,210, and 86 amu, and the close similarity of the spectra of the other homologous alkaloids (Brunner et d., 1979; Stadler et al., 19771, prompted further investigations into the identity of the ergot peptide alkaloids produced by E. typhina. Comparative TLC and low-resolution mass spectra eliminated all of the natural and synthetic alkaloids of the ergotoxine and ergoxine groups (Brunner et al., 1979) and all but ergosine and ergovaline from the ergotamine group. Interpretation of ergovaline's mass spectra (Le., electron impact, EI, at 70 eV) is complicated by the fragmentation of the tricyclic peptide moiety resulting in the same atomic mass ions as the lysergic acid amide and clavine portions (Vokoun et al., 1974; Vokoun and Rehacek, 1975) of the alkaloid. The above is further compounded by the almost identical chemical and physical properties of ergosine and ergovaline (Brunner et al., 1979) and the problems of working with small quantities of a natural isolate (Porter et al., 1979a). Chemical ionization (CI) mass spectroscopy's (Arsenault, 1972; Fales et al., 1970) compliment to the low-resolution electron impact (EI) has been reported for differentiation of the alkyl substituents attached to the tricyclic peptide moiety of these alkaloids (Porter and Betwoski, 1981). Therefore, CI mass spectroscopy was used to compare the natural peptide alkaloids from E. typhina with authentic ergosine and ergovaline. Cultures of B. epichloe and B. strangulans were reported (Bacon et al., 1979; Porter et al., 1979a,b) to produce a clavine alkaloid (M+ 240) that we now report as 6,7-secoagrocalvine. The identification was based on the comparison with a synthetic standard using ultraviolet (UV) and mass spectroscopy, cochromatography, and color reaction (blue) with p-(dimethy1amino)benzaldehyde (PDAB) and also by a comparison of the methylated derivative of the natural alkaloids with synthetic N methyl-6,7-secoagroclavine(Fehr, 1967). The 6,7-secoagroclavine has been recently reported as a natural product from C. purpurea (Horwell and Verge, 1979). EXPERIMENTAL SECTION
Organisms, culture, and alkaloid extraction, isolation, and detection methods were as previously described: B. epichloe (RRC 242), alkaloid no. 1 (Porter et al., 1978, 197913); B. strangulans (RRC 233), alkaloid fraction (Bacon et al., 1979) and alkaloid m l e 240 (Porter et al., 1978);E. typhina (RRC 238), ergot peptide alkaloids labeled ergosine and ergosinine (Porter et al., 1979a); E. typhina (RRC 238), a 20-week culture on media 104 (Porter et al., 1979a). In addition, E. typhina (RRC 238) was cultured for 12 weeks on 100 g of sorbitol, 10 g of glutamic acid, 1.0 g of yeast extract, 1.0 g of KH2P04,0.3 g of MgSO4-7H20,and 1000 mL of distilled water, and the pH adjusted to 5.6 with ",OH, and the B. hennirzgsiana
Porter et ai.
(RRC 243) alkaloid fraction obtained from Bacon et al. (1979). Chromatography. Thin-layer chromatography was performed on silica gel GF-254 according to reported procedures (Agurell, 1965; Porter et al., 1979a; Stahl, 1969) with the following solvent systems: chloroform-methanol (CM), 4:l (vfv);chloroform-dimethylamine (CDEA), 9:l (v/v) (Agurell, 1965); benzene-dimethylformamide (BDMF), 86.513.5 (v/v) (Stahl, 1969); chloroform-methanol-concentrated ammonia (CMA), 9 4 5 1 (Horwell and Verge, 1979);chloroform-methanol, 9 1 (v/v) in a saturated ammonia atmosphere (CMAtm) (Cassady et al., 1973); methylene chloride-2-propanol (MP), 3:l (v/v). Instrumental. Low-resolution electron impact mass spectra were obtained as reported (Porter et al., 1979a,b). Chemical ionization mass spectra were obtained via a direct inlet probe on a Varian Mat 44 quadrapole mass spectrometer equipped with an EI/CI source (-200 V). The ion source was maintained at a pressure of -200 pbar and operated at a temperature of -245 "C. The probe temperature was programmed at 20 "C/min from ambient to 250 "C. The fragment ions were observed between 175-200 "C. Only those ions occurring at a relative intensity greater than 1% are recorded. Isobutane was used as the reactant gas, and (perfluorotributy1)amine( m / e 219, 264, and 414 amu) was used as a calibration compound. Ultraviolet spectra were performed in methanol by using a Varian Cary 15 ultraviolet spectrometer. Infrared spectra were performed using KBr micropelleta in a Perkin-Elmer Model 457A infrared spectrometer. Nuclear magnetic resonance spectra were performed in CD30D by using a JEOL PS/PFT 100. Melting points were uncorrected and obtained with a Mettler FP5 apparatus connected to a Mettles FP52 microfurnace. Synthesis of 6,7-Secoagroclavine. N-Methyl-6,7secoagroclavine [M+ 254; mp 132-134 "C [lit. mp 136 "C (Fehr, 1967)]]was synthesized from agroclavine (Eli-Lilly) according to Bhattacharji et al. (1962) and separated from its A" isomer [M+254; mp 120 "C [lit. mp 118-120 "C (Fehr, 1967)] by preparative TLC on silica gel GF 254 by using the CM solvent system (Rf 0.57 and 0.50, respectively). The N-methyl-6,7-secoagroclavine(0.4 mM) in 2 mL of anhydrous acetone was treated with 0.7 mM of diethyl azodicarboxylate (Aldrich Chemical Co.) in 3 mL of anhydrous diethyl ether. The reaction mixture was stirred under N2 for 5 h and allowed to stand in the refrigerator overnight. The reaction mixture was extracted (3 X 3 mL) with a 2% tartaric acid solution. Extracts were combined, the pH was adjusted to 10 with ",OH, and the resulting solution was extracted with 3 times the equivalent volume of CHC13 The alkaloidal extracts were combined, treated with anhydrous Na2S04,filtered, and subjected to preparative TLC (20 X 20 cm glass plates, 0.75-mm silica gel GF-254)using the CDEA solvent system. The N-methyl-6,7-secoagroclavine(Rf 0.70; M+ 254) starting material separated from the reaction products (R, 0.60) in this system. Low-resolution mass analyses of the reaction product(s) isolated from the silica gel suggested it consisted of at least three compounds: M+ 252, m l e 237 (252 - CH,); M+ 240, m l e 225 (240 - CH,); M+ 176, m / e 131 (176 - OC2H5). The desired product (M+ 240) was isolated from this mixture by preparative TLC using the BDFA solvent system (Rf 0.37). After elution from the silica gel, this fraction was chromatographed in the CM solvent system (Rf0.23), followed by recrystallization from hexane [mp 132 "C [lit. mp 138 "C (Fehr, 1967); lit. mp 126-129 "C (Horwell and Verge, 1979)]],and the isolated compound had a UV, IR, and NMR as reported (Fehr, N
Ergot Alkaloid Identification in Fungi of Grasses
J. Agric. Food Chem.. Voi. 29, No. 3, 1981 655
1967). Also isolated in this chromatography system was a fraction ( R j0.57) that was visually observed as a dark blue band under 254 and 366 nm and gave a blue reaction to PDAB. Its absorption (Xmw 317 Hand 236 nm) along with the low-resolution mass spectrum (M' 252, m / e 237, 221,206,196,181,167,154, and 85) gave indications that this fraction consisted mainly of a dehydrogenated Nmethyl-6,7-secoagroclavinewith the newly formed double bond being conjugated with the indole nucleus (Cassady et al., 1973). The mass spectrum also revealed the presence of diethyl hydrazincdicarboxylate (Fieser and Fieser, 1967; Yoneda et al., 1966), M+ 176, m l e 131 and 130 amu, as a minor constituent. Methylation of Natural Products (Mt 240) Isolated from B . epichloii!and B . strangulans. Methanol, 200 pL, and 10 drops of methyl iodide (Aldrich Chemical Co.) were added to sufficient chromatography material ( 250 pg) of each natural alkaloid. The systems were flushed with N2,capped, and allowed to stand for 45 min at 40-45 "C with periodic manual agitation. The reaction mixtures were concentrated under a stream of N2to 100 pL, and each was subjected to preparative TLC in CDEA as above. This system separated starting material from the semisynthetic product (M' 254), indicative of the addition of one methyl substitutent. Festuclavine from E . typhina (Sorbitol-Glutamic Acid Culture Media). The alkaloid fraction (Porter et al., 1979a) from a 12-week culture was chromatographed on silica gel by using CM and was visualized by spraying with PDAB. The one band corresponding to a blue reaction for ergot alkaloids was scrapped from the TLC plate, eluted from the silica with chloroform-methanol, 1:l (v/v), and chromatographed with authentic pyro-, costa-, and festuclavine in solvent systems CDEA and BDMF. The natural alkaloid from this culture corresponded to festuclavine as determined by UV, TLC, and m / e (Agurell, 1965; Vokoun et al., 1974). Clavine Alkaloids from B . henningsiana. The alkaloid fraction (625 pg, based on ergonovine maleate) isolated from a 5-week culture of B. henningsiana (Bacon et al., 1979) was subjected to preparative TLC in solvent system CM and resulted in three fractions ( R j0.10,0.18, and 0.65) that gave a blue reaction with PDAB. Although fraction 1 (Rf 0.10) corresponded to chanoclavine I in CDEA (Agurell, 1965) and CMAtm (Cassady et al., 1973; Porter et al., 1978) and its low-resolution mass spectrum showed Mt 256, mle 237,183, and 154 as reported (Vokoun et al., 1974),the quantity of material isolated was not sufficient to unequivocally eliminate the isomeric relatives. Fraction 2 (Rf 0.18) cochromatographed with authentic dihydroelymoclavine (dihydrolysergol I, Eli Lilly) in CM (Rf0.18), CDEA (Rj0.15), CMAtm (Rf0.47), and MP ( R j 0.04). Comparison of the low-resolution mass spectra of this fraction and dihydroelymoclavine (Table I) conducted under identical conditions showed discrepancies which may be the results of compound purity or an isomer that will not separate by chromatography under the conditions described. Attempts to compare the acetylation material of fraction 2 with 0-acetyldihydroelymoclavinesynthesized according to Agurell et al. (1963) were unsuccessful, probably because of the quantity of the compound ori inally isolated. Fraction 3 (CM, Rf 0.65) had a X,,cH3gH and low-resolution mass spectrum (Figure 1)suggestive of either an isomer of dihydrolyseramide [CM, Rf 0.26 [lit. Rf 0.23 (Agurell, 1965)]] or a derivative of this compound, i.e., RCONHR' for R = ergoline [cf. m / e 144, Figure 1 (Barber et al., 1965; Schmidt et al., 1978)] with the R' unknown. Fermentation conditions for B. henningsiana
Table I. Mass Fragmentation Comparison of Dihydroelymoclavineand Alkaloid Fraction 2 from B. henningsiana (cf. the Text) 96
N
m le
standard
natural product
257 (M t 1) 256 (M) 24 1 23 9 23 7 225 223 21 1 209 207 197 195 194 182 168 167 159 156 155 154 153 144 127 115
15 68 5 2 3 8 16 2 6 5 24 7 9 15 26 35 1 14 31 100 13 39 36 13
15 55 5 7 7 13 15 15 13 16 25 10 16 22 27 39 16 17 36 100 22 39 45 21
1
a.
7
180
203
220
240
180
280
2 s ~
eu
80
1M
120
0 1.
Ieu
We
180
b.
7
2m
220
2.0
280
280
Figure 1.
(a) Alkaloid (fraction 3) from B. henningsiana (cf. the text); (b) dihydrolyseramide.
and the other systemic fungi mentioned above are under investigation in order to provide material sufficient for absolute identification of these alkaloids.
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Porter et ai.
Table 11. Major Diagnostic Mass Fragments of Synthetic (syn.) 6,'l-Secoagroclavine (Fehr, 1967) and Alkaloid(s) Isolated from Systemic Fungi B. epichloe(B.e.), B. strangulans (B.s.), and E. typhina (E.t.)
'"I
J
70
% m/e
240 225 208 197 194 184 182 16 8 155 127 115 85 42
syn. 100 10
16 22 37 59 10 56 17
B.e. 100 12 12 15 20 25 23 42 58 18 18 75
B.s. 100 10 12 18 21 28 26 45 79 26 20 79
E. t. 100 17 17 37 27 32 46 54 82 37 15 85 29
RESULTS A N D DISCUSSION Alkaloid Workup of 20-Week Culture of E. typhina (RRC 23858)on M 104. Preparative chromatography of the alkaloid fraction from Porter et al. (1979a) on silica gel GF 254 using CM resulted in six fractions that cochromatographed (Rf)with (1)ergosinine (0.74), (2) ergosine (0.60), (3) agroclavine (0.35), (4) penniclavine (0.26), ( 5 ) elymoclavine (0.21), and (6) chanoclavine(s1(0.09).After elution from the silica gel, these individual fractions chromatographed as homogeneous spots in the CM system with the authentic standards listed. Three of these fractions separated into additional compounds on chromatography in the CDEA solvent system. Fraction 1separated into the alkaloid that cochromatographed with ergosinine and a minor compound that was visualized as a blue spot only after spraying with PDAB. This minor compound (Rf0.76) was separated from ergosinine by developing the plates 2 times in CDEA and allowing the plates to air dry between runs. Although this minor component appeared homogeneous in CM (Rf0.74), CDEA (R 0.54), and BDMF (Rf 0.79) and its W spectrum s gested it was a simple indole or clavine alkaloid (XmCHaO Y f 292,281,273, and 224 nm), the low-resolution mass analyses of this fraction suggested a complex mixture of several compounds. As the probe temperature of the mass spectrometer was increased in 50 "C increments from ambient to 250 "C, major ions were observed at 100 "C ( m / e 244,153, 125,91, and 70 amu) and 250 "C ( m / e 369,314,297,270,255,237,225,207,194, 192, 181, 168, 167, 154, 143, and 127 amu), respectively. The above spectra may represent two compounds, one of which may be (or contain) the phenylalanylproline lactam moiety (Groger et al., 1975);also,the observed spectra may represent one compound that is subject to pyrolyses prior to electron impact similar to the ergot cyclol alkaloids (Porter and Betowski, 1981). This fraction was stored under N2 (0 "C) for future investigations. When subjected to preparative chromatography in the CDEA system, fraction 5 separated into elymoclavine (Porter et al., 197913) and another compound that corresponded [UV; TLC; m / e (Table II)] to the synthetic 6,7secoagroclavine. Fraction 6 in the CDEA system consisted mainly of chanoclavine I (W;TLC; m / e ) and a few minor compounds that were not investigated. The natural alkaloids [M+ 240 (Table II)] from B. epichloe (Porter et al., l978,1979b), B. strangulans (Bacon et al., 1979; Porter et al., 19781, and E. typhina had identical chromatography behavior with synthetic 6,7-secoagroclavine in the CM (Rf 0.21), CDEA (Rf 0.57), and CMA systems with Rf values relative to those of elymoclavine and agroclavine (Horwell and Verge, 1979).
L f
154
I -
704
?? '.
0-
\4. -
154 -
Ergot AikaioM Identification in Fungi of
Grasses
typhina as ergovaline. The above ions for the natural product (Figure 2c) are also supported by the abundant lysergic acid amide fragment at m / e 268 (61%). The CI spectrum of natural ergovaline (i.e., from C. purpurea) is compared with the spectrum of the peptide alkaloid isolated from E. typhina (parts b and c of Figure 2, respectively). The minor differences observed in the relative intensities of mle 267 and 197 between the two compounds may be a reflection of both instrument temperature and pressure at which pyrolysis and subsequent fragmentation occurs (Porter and Betowski, 1981) and possibly concentration of epimeric species. Figure 2b is the CI spectrum of ergovaline, whereas Figure 2c represents the spectrum of the epimeric mixture (i.e., ergovaline-ergovalinine) as determined by TLC. The above natural standard (Figure 2b) was isolated from a fraction containing ergosine (Brunner et al., 1979), and thus it is unknown at present if the minor fragments occurring at m / e 281 and 211 in both spectra (Figure 2b,c) represents trace amounts of ergosine (Figure 2a). Pure synthetic ergovaline was unavailable for these comparisons. Thus, the possibility exists that E. typhina produces ergosine and ergosinine (Porter et al., 1979a) in trace amounts with ergovaline and ergovalinine as the major components. The above comparisons do not eliminate the possibility of the peptide alkaloids from E. typhina as being isomeric relatives of ergovaline and ergovalinine. Conformational analyses of these alkaloids, as with the clavine alkaloid from Balansia, are predicated on quantities sufficient for these determinations. Although the synthetic peptide alkaloids ergovaline and ergovalinine have been known for some time (Stadler et al., 1964),only recently have these alkaloids been reported as natural products (i.e., from cultures of C. purpurea) (Brunner et al., 1979). ACKNOWLEDGMENT We thank P. A. Stadler, Sandoz Ltd., Basel, Switzerland, for the ergot peptide alkaloids ergosine and ergovaliie used in this study. We also thank H. G. Floss, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN, for festuclavine, pyroclavine, and costaclavine and for information and suggestions on the identification of 6,7-secoagroclavine. We thank E. C. Kornfeld, Eli-Lilly, Indianapolis, IN, for agroclavine, dihydroelymoclavine, and dihydrolysergamide. We thank John S. Robbins, Rita M. Bennett, Clark H. Groover, and J. C. Herndon for their technical assistance during the
J. Agric. Food Chem., Vol. 29, No. 3, 1981
857
c o m e of this work and Linda Smith, Tobacco and Health Laboratory, R. B. Russell Research Center, for low-resolution mass data. LITERATURE CITED Agurell, S. Acta. Pharm. Suec. 1965,2, 357. Agurell, S.; Ramstad, E.; Ullstrup, A. Plunta Med. 1963,11,392. Arsenault, G. P. In “Biochemical Applications of Mass Spectrometry”; Waller, G. R., Ed.; Wiley-Interscience: New York, 1972. Bacon, C. W.; Porter, J. K.; Robbins,J. D. J. Cen. Microbiol. 1979, 113, 119.
Barber, M.; Weisbach, J. A,; Douglas, B.; Dudek, G. 0. Chem. Ind. (London) 1965, 1072.
Bhattacherji, A.; Birch, A. J.; Brack, A.; Hofmann, A.; Kobel, H.; Smith, H.; Winter, J. J. Chem. SOC.1962, 421. Brunner, R.; Stiitz, P. L.; Tscherter, H.; Stadler, P. A. Can. J. Chem. 1979,57, 1638.
Cassady, J. M.; Abou-Chaar, C. I.; Floss, H. G. Lloydia 1973,36, 390.
Fales, H. M.; Lloyd, H. A.; Milne, G. W. A. J. Am. Chem. SOC. 1970,92, 1590.
Fehr, T. Dissertation No. 3967, ETH, Ziirich, 1967. Fieser, L. F.; Fieser, M. “Reagents in Organic Synthesis”;Wiley: New York, 1967; Vol. I, p 247. Groger, D.; Syring, U.; Johne, S. Pharmazie 1975, 30, 440. Horwell, D. C.; Verge, J. P. Phytochemistry 1979, 18, 519. Porter, J. K.; Bacon, C. W.; Robbins, J. D. Lloydia 1978,41,654. Porter, J. K.; Bacon, C. W.; Robbins, J. D. J.Agric. Food Chem. 1979a, 27,595.
Porter, J. K.; Bacon, C.W.; Robbins, J. D. J. Nat. Prod. 1979b, 42, 309, 696.
Porter, J. K.; Betowski, D. J. Agric. Food Chem. 1981, preceding paper in this issue. Schmidt, J.; Kraft, R.; Voigt, D. Biomed. Mass Spectrom. 1978, 5, 674.
Stadler, P. A.; Frey, A. J.; Ott, H.; Hofmann Helu. Chim. Acta 1964,47,1911.
Stadler, P. A,; Stutz, P.; Sturmer, E. Erperientia 1977,33, 1552. Stahl,E. ‘Thin Layer Chromatography,a Laboratory Handbook”; Springer-Verlag: New York, 1969; No. 74, p 452. Vokoun, J.; Rehacek, Z. Collect. Czech. Chem. Commun. 1975, 40, 1731.
Vokoun, J.; Sajdl, P.; Rehacek, Z. Zentralbl. Bakteriol., Parasitenkd., Znfektionskr. Hyg., Abt. 2, Naturwiss.: A&., Landwirtsch. Tech. Mikrobiol. 1974, 129, 499. Yoneda, F.; Suzuki, K.; Nitta, Y. J. Am. Chem. SOC.1966,88,2328.
Received for review August 4,1980. Accepted February 9,1981. Presented in part at the 20th annual meeting of the Americal Society of Pharmacognosy,Purdue University, West Lafayette, IN, July 29-Aug 3, 1979.
